This collaborative proposal from a cardiac molecular geneticist and a RNA-chromatin biochemist will investigate chromatin associated and transcription factor dependent non-coding RNAs (ncRNAs) as essential components of a cardiac regulatory network. We have applied a novel approach for identification of functional long non-coding RNAs (lncRNAs) and the enhancers that comprise gene regulatory networks. Using transcription factor- dependence, we identified ncRNAs as markers of cis-regulatory elements essential for a mouse cardiac rhythm gene regulatory network. This approach identified exceptionally strong enhancers, and their associated ncRNA are chromatin-bound and required for enhancer function. This proof of principal sets the stage for the investigation of lncRNAs and their associated enhancers in human cardiac gene regulatory networks. We posit that application of our approach to human cardiomyocytes will allow identification of a functional class of human heart enhancer-associated lncRNAs and cis-regulatory elements (CREs) essential for the expression of human cardiac rhythm control genes. In the first specific aim, we will interrogate a set of lncRNAs defined by their TF- dependence and chromatin localization for their requirement for CRE activity and target gene expression. In the second aim, we will utilize novel molecular tools to probe the molecular mechanisms whereby the lncRNAs modulate gene expression. Together these interconnected aims rely on the complementary and non-overlapping expertise of the collaborative investigators to address a highly significant problem in ncRNA biology. These hypotheses are applicable to human genetics, transcriptional regulation, and RNA biology, and therefore may have impact both within cardiovascular genetics and more broadly within human molecular genetics.